A high throughput screen of the Pfizer compound collection was carried out using a hepatitis C virus (HCV) genotype 1b subgenomic replicon cell line. Those confirmed hits that demonstrated broad spectrum activity without overt cytotoxicity were further evaluated, leading to the identification of a series of pyrrolopyridines with excellent antiviral activity in a fully infectious HCV cell-based assay and pharmacokinetic properties.
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 Genotype 1b (Con1, Licensed from Reblikon GmbH) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105 Cells/ml by Addition of Pre-Warmed Medium (DMEM + 10% FCS). 45 μl of This Suspension Was Added to Each Well a 384-Well Assay Plate (Lumitrac, Greiner) Already Containing 0.5 l of Test Compound. All Plates Were Covered with gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, the Plate Was Removed from the Incubator and Left to Cool to Room Temperature for 15 -30 mins. An Equal Volume of Reconstituted Lyophilised Britelite plus Substrate (PerkinElmer) to Medium Was Added to Each Well. Luminescence Was Read Immediately on an EnVision (Perkin Elmer) Plate Reader.
 1[2-(4-Iodophenyl)-3-(4-Nitrophenyl)-5-(2,4-Disulfophenyl)-2H-Tetrazolium], CAS Registry Number, 150849-52-8.
 Genotype 1b (Con1) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105cells/ml by Addition of Pre-Warmed Medium (DMEM +10% FCS). 45 μl of This Suspension Was Added to Each Well of a 384-Well Assay Plate (Greiner) Already Containing 0.5 l of Test Compound. All Plates Were Covered with Gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, 5 μl of WST-1 Cell Proliferation Reagent (Roche) Was Added to Each Well and the Plate Returned to the Incubator for 1 Hour. After This Incubation Period Absorbance Was Read at 450 nm on an EnVision (Perkin Elmer) Plate Reader.
 Genotype 1a (H77, Licensed from Apath LLC) HCV Replicon Cells Were Resuspended to a Concentration of 1.4 × 105 Cells/ml by Addition of Pre-Warmed Assay Medium (DMEM + 10% FCS). 45 μl of This Suspension Was Added to Each Well of a 384-Well Assay Plate (Lumitrac, Greiner), Already Containing 0.5 l of Test Compound. All Plates Were Covered with Gas Permeable Seals and Incubated at 37oC, 5% CO2 for 48 Hours. After 48 Hours, the Assay Plate Was Removed from the Incubator and Left to Cool to Room Temperature for 15-30 mins. Medium Was Removed from the Wells and 5 μl Lysis Buffer (Renilla Luciferase Assay Kit, Promega) Was Added to Each Well. The Plate Was Incubated at Room Temperature on a Rocker for 15 mins then 15 μl Assay Substrate was Added to Each Well. Luminescence Was Read Immediately Using an EnVision Plate Reader.
 Details of the Biochemical Assays and compound Screening within Them Will Be Described Elsewhere.
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 5-(2-Thienyl)-N-(2-Thienylmethyl)-7-(Trifluoromethyl)-pyrAzolo[1,5-a]Pyrimidine-2-Carboxamide, CAS Registry Number 314049-21-3.
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